Alzheimer's disease (AD) is the most common neurodegenerative disorder, affecting over 5 million Americans. Despite efforts to develop AD therapies, those that are FDA approved do not effectively slow neurodegeneration nor disease progression. The focus of our studies is to discover novel drugs targeting the toxic actions of apolipoprotein (apo) E4 to block neurodegeneration in AD. ApoE4 is the major genetic risk factor for AD, and apoE4 carriers account for 65-80% of all cases of AD. ApoE4 increases the occurrence and lowers the age of onset of AD, and considerable evidence suggests that it has a fundamental role in AD neurodegeneration. We propose to identify small-molecule probes that can correct the pathological conformation of apoE4 (structure correctors), abolish its neurotoxic effects, and potentially serve as new drug leads to treat or prevent AD progression. Two major isoforms of apoE, apoE3 and apoE4, are expressed in the brain. They differ by one amino acid, and that single substitution (cysteine to arginine) converts apoE3 from a molecule that supports neuronal maintenance, promotes neurite outgrowth and neural repair, and protects neurons to a neuropathological form (apoE4). This amino acid difference gives rise to profound differences in the tertiary protein structure and function. ApoE4 displays an intramolecular domain interaction between its amino- and carboxyl-terminal domains, leading to a compact structure. Disrupting apoE4 domain interaction with small-molecule structure correctors converts apoE4 into an apoE3-like conformation and reverses the apoE4-specific detrimental effects on neurons. In pilot screens, we found small-molecule structure correctors that disrupt apoE4 domain interaction, prevent the formation of toxic apoE4 fragments, and protect mitochondria and neurons from degenerating. The objective of the current proposal is to identify novel chemical series of apoE4 structure correctors that can be developed into chemical probe(s) with improved potency and pharmaceutical properties that will potentially lead to a new generation of drugs to treat AD. To achieve this goal, we developed cell-based high-throughput screening assays to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify small-molecule structure correctors of apoE4. A primary assay will measure green fluorescent protein (GFP)-apoE4 levels. A secondary assay based on GFP- apoE4-eDHFR fluorescence resonance energy transfer will be used to confirm hits selectively. In addition, tertiary cell-based assays will measure the toxic effects of apoE4 on cytochrome c oxidase subunit 1 of complex IV and measure neurite outgrowth. These final assays will further validate the specificity of action and biological relevance of the candidate compounds in protecting mitochondrial function and preventing neuronal degeneration. Probes identified from these studies can be used to further study the role of apoE4 in AD and will serve as a foundation for the development of novel drugs to treat AD.